Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARF) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of sARF I and II, purified from bovine brain (Tsai et al. (1988) J. Biol. Chem. 263: 1768-1772), established that they are ARF 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARF 1 and 3 were distinquished by their electrophoretic mobilities; ARF 2 was not detected in rat brain. Rabbit antiserum against rARF 5 cross-reacted partially with rARF 4, but not detectably with rARF 6 and minimally with class I ARFs. ARF 4, which differs from ARF 5 in electrophoretic mobility, was not detected in rat brain. GTPgammaS increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. Saccharomyces cerevisiae has two ARF proteins that are 96% identical. The ARF 1 gene is constitutively expressed whereas the ARF 2 gene is repressed by glucose. Expression of human ARF 5 or ARF 6 or Giardia ARF can resume much more slowly, however, than wild-type yeast or mutants rescued with yeast ARF 1, from which we infer that the heterologous ARFs may not function efficiently or effectively in the yeast vesicular protein trafficking system.